Nephrotic syndrome (NS) is a common kidney disease, but the molecular mechanisms underlying the disease remain unclear for the vast majority of cases. We previously reported that the small heat shock protein, hsp27, a known regulator of actin polymerization, is highly expressed in glomerular podocytes (the cells most affected in NS), and that glomerular hsp27 expression and phosphorylation are significantly induced in experimental NS. We also reported that hsp27 is able to dramatically regulate (i.e. hsp27 overexpression protects, while reduced expression sensitizes) the podocyte response to PAN-induced cellular injury and actin cytoskeletal disruption. More importantly, we recently confirmed that glomerular hsp27 expression is induced in both multiple animal models of NS, as well as in human NS, suggesting that induction of glomerular hsp27 represents a generalized podocyte stress response. Since hsp27 has not been confirmed to bind actin directly in vivo, we attempted to clarify the mechanism of hsp27-mediated regulation of podocyte structure by screening a glomerular yeast two-hybrid library, and identified two novel hsp27 binding proteins: 1) Hic-5, a known focal adhesion protein and paxillin homologue, and 2) Arpda, a known member of the Arp2/3 actin polymerization initiation complex. We confirmed hic-5 as a true hsp27 binding protein by co-IP, mapped its interaction domains with hsp27, and showed that hic-5 can inhibit hsp27-induced thermo-protection in an interaction-dependent manner. We also confirmed both ArpCIa and ArpClb as true hsp27 binding proteins by co-IP and quantitative FRET analyses. Based on the above, we hypothesize that hsp27 plays a critical role in the regulation of podocyte structure and response to injury, and that these actions are mediated via its novel binding proteins, hic-5 (a paxillin homologue with a role in focal adhesion dynamics), and ArpC1 (a member of the Arp2/3 actin initiation complex). To test this hypothesis we will complete development of podocyte-specific hspbl (hsp25) gene-targeted mice and perform phenotypic analyses to definitively determine the role of hsp25 in podocyte biology. We will also determine if induced alterations in hic-5 expression or interaction with hsp27 can regulate podocyte: 1) Focal adhesion formation rate, composition, and function (i.e. adhesion), and 2) Response to injury. Lastly, we will determine if induced alterations in ArpC1 expression or interaction with hsp27 can regulate podocyte: 1) Arp2/3 complex function (i.e. initiation of actin polymerization) and composition, and 2) Response to injury. Confirmation of hsp27's importance in the regulation of podocyte structure, and identification of its molecular mechanism(s) of action in podocytes, could not only improve our understanding of the molecular mechanism(s) underlying the development of NS, but also permit the development of more highly targeted and/or less toxic therapies for this very common kidney disease.